In the past year, we continued to study P. falciparum genome diversity, drug resistance, gene regulation, and virulence using genetic mapping, microarray, gene knockout, genetic crosses, population genetics, and other approaches. The ultimate goals were to relate genetic polymorphisms to parasite biology and disease and to develop novel approaches to control malaria. We have obtained thousands of single nucleotide polymorphism (SNP) from 185 culture-adapted isolates, tested the parasites responses to 7 antimalarial drugs, used the genotype/phenotype data in genome-wide association analyses, and identified candidate genes associated with parasite drug responses to mefloquine (MQ), dihydroartemisinin (DHA) and other drugs. Recently, we initiated a project to systematically characterize parasite differential response to thousands of chemical compounds and have collected hundreds of differential chemical phenotypes (DCPs). Genetic variations underlying some of these differences have been identified through genetic mapping. Gene functions can be deduced after further studies of the candidate genes. This study also identified many potential antimalarial compounds, some of which are being tested in vivo. To study virulence and disease phenotype, we are developing a genetic map for Plasmodium yoelii. Disease phenotypes are the results of parasite and host interactions. Use of inbred mice will greatly reduce the influence of host factors on phenotype measurement. We have identified 600 microsatellite (MS) markers, performed three genetic crosses using different parasite isolates, and obtained 45 progeny from the crosses. We are in the process of cloning more progeny and evaluating various phenotypes from the progeny. Several studies showed differences in gene expression between pairs of isogenic parasites, but no changes in DNA sequences could be identified, including the pfmdv 1 we described previously. One possibility for the differences in gene expression is epigenetic regulation of gene expression. Nucleosome positioning is one of the epigenetic gene regulation mechanisms. It has been shown that nucleosome phasing relative to transcription start sites is directly correlated to RNA polymerase II binding, and the first nucleosome downstream of a start site exhibits differential positioning in active and silent genes. We are interested in the relationships in parasite nucleosome position, gene expression, and different parasite developmental stages and have obtained nucelosome positions from different developmental stages.